The long-term goal of this project is to investigate functions of gap junctional coupling in the inner ear. A gap junctional channel is an intercellular cytoplasmic conduit, and is encoded by the connexin gene family in mammals. Six connexin isoforms assemble to form a hexadal connexon (hemichannel), and dock with another connexon in the opposite cell membrane to form an intercellular gap junctional channel through which molecules up to 1 kDa can pass. Cx26 and 30 are the predominant isoforms in cochlear supporting cells. Each connexin mutation can induce hearing loss. It has been found that gap junctions between supporting cells have various asymmetrical voltage gates, indicative of the occurrence of heterotypic and heteromeric hybrid channel configurations in the cochlea. The working hypothesis of this project is that Cx26 and 30 may dock to form heterotypic and heteromeric channels with specific permeability to selectively transfer ions and molecules in the cochlea; asymmetric heterotypic channels may also induce directional intercellular transfer in this multicellular system. Specific aim (SA) 1 is to define connexin-specific functions in the cochlea. We will use immunofluorescent staining to identify connexin expression and distributions, patch clamp recording to measure channel conductance and gating to identify channel types and configurations, and fluorescent probes to assess connexin channel permeability. Charge and size selectivity in gap junctional permeability will be assessed by use of multiple florescent probes with patch clamp recording. Fluorescence recovery after photo bleaching (FRAP) with confocal microscopy will also be employed to quantitatively define gap junctional permeability. The asymmetry of transjunctional diffusion between cochlear supporting cells and directional passage in the cochlear sensory epithelium will be defined by FRAP measurement and time-lapse fluorescent microscopy (SA2). SA3 is to define the activity and permeability of connexin hemi channels in native cochlear supporting cells to further elucidate structure-function relationship of gap junctional coupling in the cochlea. SA 4 is to test the K+-recycling hypothesis that has long been proposed but has yet to be tested. We will use patch clamp technique to directly record K+ passage between cochlear supporting cells. SA5 is to explore the effect of supporting cell's junctional coupling on outer hair cell electro motility. Undoubtedly, completion of these studies will contribute signaificantly towards understanding the mechanisms of gap junctional coupling in the inner ear, and in turn, develop therapeutic and protective interventions for this common hereditary deafness.